With the increasing interest in well-being or a healthy life, tagatose has been proposed as an alternative to sugar as it has less side effects and sugar is one of the major factors causing various adult diseases. Tagatose is the isomer of galactose and is known to have fructose-like physiochemical properties. Tagatose is a natural low-calorie sugar, and has recently been approved by the FDA in the USA as GRAS (Generally Recognized As Safe), so it is now allowed to be added as a sweetener to foods, beverages, health foods, diet additives, etc.
GRAS indicates a substance that is generally recognized as safe, which is judged by specialized people having enough experience and skills through scientific procedure and examination under the indicated conditions and purpose of use. GRAS is a unique system used only in the USA to evaluate the safety of foods and food chemical substances (under certain conditions), but it is recognized world-wide.
Tagatose is produced by either isomerization, which is a chemical method using a catalyst to produce an isomer, of galactose or a biological method using isomerase to convert galactose enzymatically.
One of the biological methods well-known to those in the art is to convert aldose or aldose derivatives into ketose or ketose derivatives by using an enzyme. The isomerization of galactose into tagatose using arabinose isomerase is generally carried out thermodynamically at high temperature and exhibits a proportionally high conversion rate. Therefore, developing an enzyme that works stably at high temperature and a method of preparing tagatose using the same are key techniques for the industrial application thereof based on the biological conversion of tagatose using an isomerase. By screening thermophile derived arabinose isomerases, an industrially applicable thermophilic isomerase has been tried, and efforts have also been made by many research teams to establish an isomerization process using the same.
In Korea, an enzymatic isomerization method using arabinose isomerase has been developed by Tong Yang Confectionery Corp. According to the method, an E. coli derived arabinose isomerase gene was mass-expressed in E. coli by recombinant technology. This recombinant isomerase was reacted at 30° C. for 24 hours to convert galactose into tagatose, and at this time the conversion rate was 25%, indicating that both thermostability and transformation yield were very low (Korean Patent Application No. 99-16118). Professor Oh and his colleagues (Sejong University) succeeded in the mass-expression of arabinose isomerase originating from Geobacillus stearothermophilus in E. coli by recombinant technology, and based on that they proposed an isomerization procedure at high temperature to convert galactose into tagatose. Tong Yang Confectionery Corp. team separated a thermophilic isomerase from a hot spring area by screening the thermophilic microorganism library and then expressed it as an active form in a recombinant E. coli host to use galactose for the high temperature isomerization process. Similarly, CheBiGen Inc. also produced thermophilic isomerase originating from Geobacillus dinitrificans DBG-A1 in E. coli and developed a technique to produce tagatose by immobilization using the same.
The expression level of the isomerase originating from the Geobacillus microorganism is too low to be applied in industry. The production of tagatose using a thermophile derived arabinose isomerase still depends on the method of using a recombinant enzyme mass-expressed in recombinant E. coli or the isomerization of galactose into tagatose using a host containing the recombinant enzyme. However, this biotechnological production of tagatose using recombinant E. coli is not appropriate for the production of tagatose as a food material. To produce tagatose as a food additive, arabinose isomerase expressed in a host which is a GRAS microorganism appropriate for the mass-production of the same is essential.
Industrially applicable GRAS microorganisms for the production of a recombinant enzyme can be selected from a group consisting of Bacillus sp., Corynebacterium sp., and Lactobacillus sp. It is easy to manipulate the genes of Bacillus sp. and Corynebacterium sp. strains for industrialization and mass-culture, and they are highly stable under various conditions. In fact, among GRAS microorganisms, mostly Bacillus sp. and Corynebacterium sp. strains have been used as a host for the production of a recombinant enzyme.
The present inventors succeeded in expressing thermophilic arabinose isomerase originating from Geobacillus sp. as an active form in GRAS strains such as Bacillus sp. and Corynebacterium sp. strains. The present inventors further established a method to induce isomerization of galactose into tagatose at high concentration by using the recombinant GRAS strains.